The basis for many molecular-biological and immunoassays, diagnostic assays and tests, among other things, include the steps of obtaining a sample suspected of containing cellular material of interest (e.g., blood, tissue, food or water samples, etc), separating out the cellular material of interest, disrupting or lysing the cells of interest to release the crude lysate (containing proteins, nucleic acids, cellular components, etc.), purifying the crude lysate (i.e. removing unwanted cellular debris), and performing some analysis on the lysate to detect the molecules or components of interest.
The current methods commonly used in biological laboratories for manipulation, concentration, and separation of bioparticles and macromolecules include optical tweezers, fluorescence or magnetic field activated cell sorting, centrifugation, filtration, and electric field-based manipulations and separations. Among these methods, the electric field based approach is well suited for miniaturization because of the relative ease of microscale generation and structuring of an electric field on microchips.
Depending on the nature of bioparticles to be manipulated, different types of electric fields can be applied: (1) a DC field for electrophoresis (EP) of charged bioparticles; (2) a nonuniform AC field for dielectrophoresis (DEP) of polarized bioparticles; (3) the combined AC and DC fields for manipulating charged and neutral bioparticles. Because most biological cells have similar electrophoretic mobilities, EP for manipulation of cells has limited applications. On the other hand, DEP has been successfully applied on microchip scales to manipulate and separate a variety of biological cells including bacteria, yeast, and mammalian cells.
Large-scale dielectrophoresis has become a popular technique for separating microparticles which are either charged or uncharged in solution. These techniques are usually performed in a glass slide based device having exposed (i.e. naked) interdigitated electrodes plated on the surface of the slide and having a flow chamber with a volume of several hundred microliters. Cells are separated in such devices based on their dielectric properties by choosing separation buffer(s) with appropriate conductivity and an AC signal with a suitable amplitude and frequency. These prior devices have several problems, including the following. A first problem is that both separated and unseparated cells bind nonspecifically to the exposed glass surface of the slide and to the exposed electrode surfaces. A second problem is that the volume of the flow chamber (several hundred μl) is so large that thermal convection disturbs and pushes off cells initially retained by the electrodes. A third problem is that washing off any undesired cells is not easily accomplished without disturbing the cells that are desirably retained on the electrodes, as the desired cells and electrodes stand in the way of fluidic flow and, hence, block the wash flow containing any undesired cells.
To separate intracellular organelles and molecular components, cells must be disrupted. Disrupting or lysing cells releases the crude DNA and RNA material along with other cellular constituents. Well known electronic cell electroporation lysing techniques are conventionally performed by applying a series of high voltage DC pulses in a macrodevice, as opposed to a microchip-based device. These conventional electronic lysis/electroporation techniques have several problems. A first problem is that the electronic lysis conditions specified by commercial macro-devices do not release medium to large proteins, organelles, and DNA molecules of high molecular weight (larger than 20 Kb) because they do not fit through the pores created in the cell membrane by the prior lysing methods. A second problem is that some molecules of interest originally released in the lysis chamber are lost due to their non-specific binding to the surface of the lysis chamber. A third problem is that the conventional electronic lysis macro-device works as a stand-alone unit such that both dielectrophoretic cell separation and electronic lysis cannot be performed on the same module.
The crude lysate is then purified (i.e., undesired cellular debris is washed off or separated), and then the purified lysate is subjected to various enzymatic reaction(s) and/or other processing steps to prepare the lysate for detection and analysis. These conventional preparation and processing techniques have several problems, including the following. A first problem is that the steps of sample preparation and processing are typically performed separately and apart from the other main steps of the analysis. In addition, most of these techniques involve carrying out numerous operations (e.g., pipetting, centrifugations, and electrophoresis) on a large number of samples. They are often complex and time consuming, and generally require a high degree of skill. Many a technique is limited in its application by a lack of sensitivity, specificity, or reproducibility.
Attempts have been made to use dielectrophoresis to separate and identify cells. For example, U.S. Pat. No. 4,326,934 to Herbert discloses a method and apparatus for cell classification by continuous dielectrophoresis. Cells were separated by making use of both the positive and negative dielectrophoretic movement of cell particles. Separated cells were allowed to be characterized and/or classified by viewing the characteristic deflection distance of cells moving through the two electrodes.
Also, U.S. Pat. No. 5,344,535 to Walter et al. discloses a method and apparatus for the characterization of microorganisms and other particles by dielectrophoresis. Cells were characterized by matching their signature dielectrophoretic collection rates. U.S. Pat. No. 5,569,367 to Walter et al. discloses a method and apparatus for separating a mixture using a pair of interdigitated electrodes. The apparatus used two energized interdigitated electrodes that obstruct straight through flow of cells and further separate different types of cells into fractions by applying a non-uniform alternating field. The electrode structure is comprised of interleaved grid-like structures aligned to obstruct flow through the structure.
In addition, attempts have been made to combine certain processing steps or substeps together. For example, various attempts have been made to describe integrated systems formed on a single chip or substrate, wherein multiple steps of an overall sample preparation and diagnostic system would be included. For example, A. Manz et al., in “Miniaturized Total Chemical Analysis System: A Novel Concept For Chemical Sensing”, Sensors And Actuators, B1 (1990), pp. 244-248, describe a ‘total chemical analysis system’ (TAS) which comprises a modular construction of a miniaturized TAS. Sampling, sample transport, any necessary chemical reactions, chromatographic separations as well as detection were to be automatically carried out.
Traditional immunoassay methods utilizing microtiter-plate formats, dipsticks, etc., are labor and time extensive. Multiple steps requiring human intervention either during the process or between processes are sub-optimal in that there is a possibility of contamination and operator error. Further, the use of multiple machines or complicated robotic systems for performing the individual processes is often prohibitive except for the largest laboratories, both in terms of the expense and physical space requirements.
As is apparent from the preceding discussion, various methods exist to provide traditional means of immunoassay analysis. However, for the reasons stated above, these traditional techniques involve the disadvantages of multiple sample/analyte transfer steps, and often require large sample volumes to obtain the desired sensitivity and specificity for the assay.